Apolipoprotein A-IV Has Bi-Functional Actions in Alcoholic Hepatitis by Regulating Hepatocyte Injury and Immune Cell Infiltration

Figures

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  Figure 1. APOA4 is elevated in AH. Volcano plots showed the differentially expressed genes (DEGs) between AH and normal liver tissues in the E-MTAB-2664 dataset and the GSE28619 dataset. Orange dots indicate the up-regulated genes, blue dots indicate down-regulated genes, and gray dots indicate non-DEGs. (A,B) Volcano map and Box plot of APOA4 expression between AH and normal liver tissues in the E-MTAB-2664 dataset. (C,D) Volcano map and Box plot of APOA4 expression between AH and normal liver tissues in the GSE28619 dataset. (E) Hematoxylin-eosin (H&E) staining. (F,G) The activity of ALT and AST. (H,I) The expression of APOA4 in AH tissue as well as serum. (J) The expression of APOA4 in AML-12 cells after 200 μmol/L ETOH treatment for 12 h. (n = 3–6) * p < 0.05, ** p < 0.01 vs. the control group.

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  Figure 2. APOA4 inhibition decreased cell injury in ETOH -treated AML-12 cells. (A) AML-12 cells were transfected with APOA4 siRNA or control siRNA for 48 h. The expression of APOA4 was determined with qRT-PCR. (B) The AST and ALT activity in AML-12 cells after treatment with APOA4-si stimulated by ETOH or left untreated. (C) IL-1β activity by IL-1β ELISA kit. (D) ROS fluorescence intensity was detected by flow cytometry. (n = 3–6) * p < 0.05, ** p < 0.01 vs. the control group; # p < 0.05 vs. the ETOH group or NS p > 0.05 vs. the Control group.

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  Figure 3. Functional enrichment and PPI analysis of APOA4 co-expressed genes. (A) A Pearson test was used to analyze the correlation between APOA4 and other genes in AH. Red indicates positively correlated genes; blue indicates negatively correlated genes. (B,C) Heat maps illustrating genes positively and negatively correlated with APOA4 in AH (top 50). (D) Analysis of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways associated with APOA4 expression. (E) Network plot of the relationships among the top 20 terms. (F) PPI network plot of genes co-expressed with APOA4.

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  Figure 4. Validation of hub genes. (A) AMBP, (B) CIDEC, (C) FETUB, (D) LCAT, (E) LPA, and (F) TTC36.

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  Figure 5. The role of CIDEC in AH. (A) CIDEC, in the ArrayExpress cohort. (B) The expression of CIDEC in AH tissue compared with the control group. (C) The correlation of CIDEC and APOA4 in AH liver tissue. The correlated concentration between APOA4 and CIDEC was determined by western blotting (D) and qRT-PCR (E). (n = 3–6) * p < 0.05, vs. the Control group.

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  Figure 6. Proportions of 22 immune infiltrating cells’ subpopulations in normal liver tissues and AH tissues were associated with APOA4. (A) Analysis of Gene Ontology associated with APOA4 expression by GSEA enrichment analysis. (B) Immune infiltrating cells in normal liver tissues. (C) Immune infiltrating cells in AH tissues. * p < 0.05, ** p < 0.01 vs. the high group, ns p > 0.05 vs the high group.